SUMMARY The loss of tissue regenerative capacity is an almost ubiquitous aspect of mammalian aging. Underlying this age-related change is a decline in the potential of tissue-specific stem cells to participate in tissue repair and regeneration. In skeletal muscle, this has been amply demonstrated for the resident muscle stem cells (MuSCs), whose response to injury has been shown to decline with age in response to cell-intrinsic changes and to suppressive activity from the aged systemic milieu, the former perhaps arising from the latter. Nevertheless, the molecular basis of the cellular changes that integrate these diverse inputs to render a MuSC less responsive with age remains to be determined. Likewise, the molecular basis for the cellular memory that persists ex vivo and that can be ?reprogrammed? in vivo in response to different environments remain to be elucidated. Understanding these fundamental mechanisms of MuSC aging provide the potential for being able to intervene to restore youthful characteristics to aged stem cells, thus enhancing aged tissue repair and regeneration. Toward these goals, this Project focuses primarily on epigenetic mechanisms based on the hypothesis that it is the epigenome that integrates diverse signals, mediates cellular responses, and is amenable to reprogramming in response to diverse environmental influences. In collaboration with Projects 2 and 3 and with Core C, we will explore the epigenetic features and regulators of young and aged MuSCs, in terms of transcriptome (RNA-seq), epigenome (ChIP-seq), and nucleosome positioning (ATAC-seq). In collaboration with Core B, we and Projects 2 and 3 will explore in more detail the notion of ?epigenetic rejuvenation? by studying the epigenetics of aged MuSCs (and other stem cells) exposed to rejuvenating interventions that have been shown to restore youthful function to aged stem cells. We will examine a specific histone mark, trimethylation of lysine 27 on histone 3 (H3K27me3) what we have shown previously to be strikingly enriched in aged MuSCs. We will examine how that pattern changes in response to rejuvenating strategies and how it is regulated by chromatin modifiers. In collaboration with Project 3, we will explore the role of DNA methylation in MuSC aging, also testing how this epigenetic pattern, and resulting cellular function, changes in response to Core B interventions and to alterations in expression of DNA demethylases. We will also use modified CRISP/cas9 technology to modify DNA methylation in a locus-specific manner. Finally, in collaboration with Project 2, we will explore the population dynamics of MuSC aging by using single cell RNA-seq analysis to evaluate changes in clonal diversity and to test for clonal expansion, a process that we will also explore independently using clonal lineage tracing strategies. We will work closely with all members of this Program to integrate our efforts to lead the field of epigenetics of stem cell aging.